Substitution of valine for histidine 265 in carbon monoxide dehydrogenase from Rhodospirillum rubrum affects activity and spectroscopic states.
In carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum, histidine 265 was replaced with valine by site-directed mutagenesis of the cooS gene. The altered form of CODH (H265V) had a low nickel content and a dramatically reduced level of catalytic activity. Although treatment with NiCl2 and CoCl2 increased the activity of H265V CODH by severalfold, activity levels remained more than 1000-fold lower than that of wild-type CODH. Histidine 265 was not essential for the formation and stability of the Fe4S4 clusters. The Km and KD for CO as well as the KD for cyanide were relatively unchanged as a result of the amino acid substitution in CODH. The time-dependent reduction of the [Fe4S4]2+ clusters by CO occurred on a time scale of hours, suggesting that, as a consequence of the mutation, a rate-limiting step had been introduced prior to the transfer of electrons from CO to the cubanes in centers B and C. EPR spectra of H265V CODH lacked the gav = 1.86 and gav = 1.87 signals characteristic of reduced forms of the active site (center C) of wild-type CODH. This indicates that the electronic properties of center C have been modified possibly by the disruption or alteration of the ligand-mediated interaction between the nickel site and Fe4S4 chromophore.
Spangler
N J
NJ
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Meyers
M R
MR
Gierke
K L
KL
Kerby
R L
RL
Roberts
G P
GP
Ludden
P W
PW
D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism; D003035:Cobalt / Q000494:pharmacology; D003486:Cyanides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009532:Nickel / Q000032:analysis / Q000494:pharmacology; D012247:Rhodospirillum rubrum / Q000201:enzymology*
D003486:Cyanides; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D003035:Cobalt; D009532:Nickel; D002248:Carbon Monoxide; D007501:Iron; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase